,A,f;(le

Wound healing properties of Henna leavesD M Sakarkar1*, U M Sakarkar, V N Shrikhande1, J v Vyas1, S Mandavgade1, S BJaiswaP and R N Purohit

IVidyabharatiCollege of Pharmacy, Camp, Amravati - 444 602, Maharashtra

2ChaitanyaAyurvedicMedical College, Sakegaon, Post. Bhusawal, Dist.Jalgaon, Maharashtra3Gurunanak Technical Institute, Bezonbagh, Kadbi Chowk, Nagpur, Maharashtra

4AnuradhaCollege of Pharmacy, Chikhali, Dist. Buldhana, Maharashtra

*Correspondent author, Sakarkar Building, Namuna Lane - 2, Amravati-444 601, MaharashtraE-mail: dsakarkar@rediffmail.com

Abstract

Wound healing potential of different extracts of henna leaves i.e.Lawsonia inermis Linn. was evaluated on the rat excision and incision wound

models. Lawsone isolated from the leaves was also screened for same

pharmacological activity.It was observed that the oral administration as well astopical application of ethanol extract of henna leaves and lawsone exhibited

significanthealing response in both the wound models. Further, it was found that

the topical application of ethanol extract as well as isolated lawsone was more

'effectivethan the same givenbythe oral route. Thus, topical application of ethanolextract can be successfully formulated for the wound healing activity.

Keywords: Lawsonia inermis, Mehendi, Henna leaves, Lawsone, Ethanolextract, Wound healing.

IPC Code; Int.cl,7 -A61P 17/02, C09B61/00

Experimental Work

Henna leaves were collected

from Chikhaldara forest area in Amravati

district, during September 2002 and were

authenticated at Chaitanya Ayurvedic

Medical College, Sakegaon, Bhusawal.

Henna leaves

The present study was aimed·.to

investigate the possible effect of differentextracts of the leaves of henna and its

formulations. The effectof the extracts on

wound repair was also investigated after

administering these extracts through oral

and topical routes. Thephytochemical and

pharm~cological action of the plant drug

through transdermal drug deliverysystem

on wound healing properties in rats was

also investigated.

Henna powder

Henna is reported to contain a

naphthaquinone, lawsone, which is a

natural dye and mainly responsible for

colouring the hair and skin of handsand feet. Lawsone has also been reportedto be an immunostimulant6• One of the

main constituents of henna leaves is

tlavonoids7,8.

Wound may be defined as a loss

or breaking of cellular and anatomic orfunctional continuity of living tissues. In

the traditional systems of medicine,various plants have been used to promote

wound healing1,2. The leaves ofLawsonia inermis Linn. syn. L. albaLam. (Family - Lythraceae),commonly called as henna (Hindi ­Mehendi) are used in the form of adecoction or ointment in the treatment of

burns, skin inflammations, wounds and

ulcers3, 4. The leaves also possess

antifungal and antibacterial activitiess.

Introduction

o ,406 Natural Product Radiance Vol 3(6) November-December 2004

Successive solvent

extraction - Air dried leaves (750g)

of henna were reduced to a fine powder,

which was subjected to hot continuous

extraction in a Soxhlet extractor,

successively with petroleum ether(40-60°C), chloroform and ethanol

(95%). Finallythe powdered material wasmacerated with chloroform water for 24

hours to obtain the aqueous extract. Each

time before extracting with next solvent,the powdered material was dried iIi' hotair oven below 50°C. Each extract was

then concentrated by distilling off the

solventfollowed byevaporation to dryness

on water bath. All extracts were kept in

desiccator and stored in refrigerator for

phytochemical and pharmacological

studies. Each extract was subjected to

qualitative chemical investigation for the

identification of phytoconstituents such as

sterols, glycosides, saponins,

carbohydrates, alkaloids, flavonoids,

tannins and proteins.

Isolation of lawsone9 ­

About 75g of crushed fresh henna leaves

were extracted by agitation for 2 hourswith 200 ml of 20% sodium bicarbonate

solution. The extract was filtered, marc

Petroleum ether (40-60°) extract

Chloroform extract

Ethanol extract

Aqueous extract

Lawsone

was again extracted with 100 ml of samesolution for 1 hour, filtered and the

alkaline extracts were pooled together.The extract was acidified with dilute

sulphuric acid and crude productobtained on standing was reextracted with

sufficient quantity of ammonium

hydroxide and again acidified with dilute

hydrochloric acid. Theproduct was finally

extracted with two successive quantitiesof benzene (40 ml) and filtered. The

filtrate was distilled to yield yellowish­

brown coloured crystals of lawsone

(mp192-193°C). Further it was

characterized by UV and IR spectralanalysis.

Preparation of ointments-The simpleointment base10 was chosen

as a vehicle for the topical application of,the extract and lawsone. Lawsone

(lOOmg) were mixed homogeneouslywith simple ointment base USP (1OOg)togetlawsoneointment (0.1% w/w). Ethanol

extract ointment (30% w/w) waspreparedby incorporating 30g of dried, powdered

ethanol extract in 100g of simple ointmentbase USPfor topical use.

,Pharmacologicalscreening- Mice of either sex of 90-days age,

Table 1: Acute toxicity studies

LD,.. (mWkg)

2200±101

1995±66

2200±101

2600±139

500

weighing 20-25g were used for acute

toxicity studies. Wistar rats of either sex

weighing 150-200g were selected for the

pharmacological study. Animal EthicsCommittee of the Institution (CPCSEA

Registration No.751) approved the studyprotocols.

Acute toxicity study - The

method suggested by Miller and Tainter(1944) 11 was used for determination of

lethal dose (LD50)' Gum acacia (1%) wasused as a vehicle to suspend the various

extracts and the suspension was

administered orally.One tenth of the LD50

was used as the maximum dose to test the

pharmacological effects possessed by theextractl2, 13 shown in Table 1.

Wound healing studies ­Wistar rats were divided into nine groups

consisting six in each and were labeled

alphabetically from A to I. Animals weredepilated at the desired site before

wounding. Theywere housed individually

with food and water given ad libitum,the basal food intake and body weights to

the nearest gram were noted. The animals

were starved for 12 hours prior to study.Excision wound - It was

inflicted on rats and the procedure was

220±10

199.5±6.6

220±10

26o±14

50

Natural Product Radiance Vol 3(6) November-December 2004 407-

A,f;,le

Results and Discussion

ethanol extract and lawsone, respectively,

applied topically once a day.

Statistical analysis -All the

results were analyzed by Student's t-test

and were expressed as mean ± standarderror of mean values. The level of

significance was set at p<O.OS.

The breaking strength in incision

wound model was evaluatedbycontinuous

constant water flow technique. All five

extracts showed increased mean breaking

strength, compared to control

(125.0± 12.1 g), when given orally once

a day. The maximum breaking strength

was seen in groups treated with lawsone

(216.0±12.5 g) and ethanol extract

(213.0±12.63 g), which were statistically

The average per cent yield of significant (p<O.OS) from control. Thepetroleum ether, chloroform, ethanol and results are shown in Table 3.

aqueous extracts were 3.93, 1.47, 31.0 The results of pharmacological

and S.31 w/w, respectively. Flavonoids, screening indicated that ethanol extract

lawsone, tannins were found to be present and lawsone have greatly contributed

in ethanol extract ind steroids, saponins towards wound healing activity(p<0.05),

in various other extracts as observed by when given orally. Therefore, in view ofthe qualitative chemical tests. The ease of treatment and comparing the

assessment of the wound healing activity efficacyof oral and topical administration,of henna, rat excision and incision wound both these extracts were studied in

mod~ls were selected and per cent wound excision and incision wound models by

closure, time to epithelization, size of scar applying_them topically in the form ofarea and breaking strength ofwound were ointment.

studied. 1\voroutes of drug administration Topical treatment - When

(oral and topical) were compared to compared to control (76.5±1.2%) on the

evaluate efficacyin wound repair. days 12, ethanol extract (98.3±0.SO%)Oral treatment-The results and lawsone (99.2±O.lS%) showed

were compared to control (86.4±1.8%) significant improvement (p<O.OS) in per

on the day 12. Lawsone (9S.40±0.6S%), cent wound closure when applied

petroleum ether extract and aqueous . topically (Table 3). Significant decrease

extract of henna showed significantly in time of epithelization (p<O.OS) was

better (p<O.OS)wound closure. Asimilar observed in groups treated withimprovement in wound closure was seen ethanol extract of henna and lawsone

with ethanol extract (96.3±O.S3%) ointment. The ethanol extract and

(p<O.OS) compared to control group. On lawsone showed complete epithelization

the other hand, the chloroform extract on 16.4±0.4S and 17.8±0.36 days,

(91.0± 1.39%) failed to show any respectively when compared to

significant difference from the control control (22.S±0.60 days) (Table 2).

(86. 4± 1.80%) (Table 2). Xhese' results Thissignifiesbetter wound healing activity.indicate that ethanol extract and lawsone The least scar areas were observed for

showed complete epithelization in ethanol extract (12.3±O.66 mm2)

18.8±0.49 and 18.6±0.82 days, followed by lawsone (14.5±1.42 mm2)

respectively (Table 2). which indicated a significant

carried out as per the method described

byMorton and Malone14• Acircular woundof about 2.5cm diameter was made on

depilated ethanol-sterilized dorsal

thoracic region of rats under light ether

anaesthesia and observed throughout the

study.The observations of per cent woundclosure were made on day 4, 8 and 12

post wounding and also for epithelizationperiod and size of scar area.

Resutured incision - The

method suggested by Ehrlich and Hunt15

was adopted. Under light ether

anaesthesia, two paravertebral incisions

of 6 cm were made through the entirethickness of the skin, on either side of thevertebral column of albino rats with the

help of a sharp blade. The incisions were

sutured using· 4-0 size silk threads with

the help of straight round-bodied needle.

On the eighth post-wounding day, sutureswere removed and the breaking strength

was determined on 10th post wounding

day by continuous constant water flow

technique of Lee16,

Drug treatment - Group A,

served as a control group and received

single daily dose of vehicle (gum acacia

1%) orally. Groups B, C, D, E and F weretreated groups and animals in these

groups received a single daily oral dose

of petroleum ether extract (220± lOmg/kg), chloroform extract (199.S±6.6mg/

kg), ethanol extract, (220±10mg/kg)

aqueous extract (260± 14 mg/kg) and

lawsone (SO mglkg), respectively. For

topical application group G, served as a

control group and 100 mg of vehicle

(simple ointment base USP), applied

topically once a day. Group H and Iwere

treated groups and received application

of 100 mg in quantity of the ointment of

Natural Product Radiance Vol 3(6) November-December 2004

L

improvement (p<O.05) when comparedto control (38.0±2.10 mm2). The results

are presented in Table 2.

The maximum means breaking

strength was seen in group treated with

ethanol extract ointment (445.0±7.5 g)

and followed by the group treated with

lawsone ointment (414.0±6.39 g) (Table3). These results substantiate the wound

healing activity (p<O.05) for the ethanolextract of henna, when compared to

control (126.o±6.3g).

The results obtained by oral

administration and topical application of

ethanol extract and lawsone were

compared with each other. The topicalroute of ethanol extract of henna and

lawsone was found to significantly

(p<O.05) promote the wound healing

activity when compared to the ethanol

extract and lawsone given by oral route

(Tables 2 and 3). Even in comparison

between topical application ethanolextract of henna and lawsone in the form

of ointment, the ethanol extract was found

effective(p<O.05) in promotion ofwound

healing than the lawsone ointment. Theresults are summarized in Table 4.

______ n n_ -----------

The phytochemical investigation

of ethanol extract revealed the presenceof both tlavonoids and the lawsone. The

tlavonoids are well-known for their role

in wound healing17• Moreover, there are

the reports that bioflavonoids have

pharmacological activities such asantimicrobiall8 and antioxidant activitiesl9•

Lawsoneis a polyphenolic compound and

is present in ethanol extract. The earlier

reports on such polyphenols revealed

significant healing effect in both wound

models used in this study20.

Table 2 :Effect of the extracts of Lawsonia inermis on the excision wound parameters--GroupCode% Wound contraction onPeriod ofMean size

givenepithelizationof scar

'Day 4DaySDay 12(days) area (mm2).----.-.--

--Oral Treatment

Control

A47.583.486.424.0 31.6±2.10

±1.66±1.86±0.45 ±1.57Petroleum ether

B59.591.695.519.5 . 22.7

(40-60°) extract

±1.20*±1.72*±1.82*±0.65*±1.79*CWorofonn extract

C56.383.391.020.5 25.3

±2.46±2.70±1.39±0.92 ±1.68

Ethanol extract

D61.893.597.818.8 10.5

±2.55*

±2.30*±0.53*±0.49* ±1.98*

Aqueous extract

E54.986.795.120.4 23.8±1.42*

±1.58±1.24*±0.72* ±1.85

Lawsone

F59.491.295.418.6 10.7

±2.57*

±1.48*±0.65*±0.82* ±1.l8*

Topical Treatment Control (simple

G42.775.376.5022.6 38.0

ointment base USP)

±1.24±1.25±1.2±0.50 ±2.81

f Ethanol extractH

41.694.898.3016.4 12.30ointment (30% w/w)±4.77

±0.67*±0.50*±0.45* ±0.66*

Lawsone ointment

I38.496.199.217.8 14.5

(0.1% w/w)

±1.24±0.62*±0.15*±0.36* ±1.42*

I Allvalues are given in Mean±S.E.; *p<0.05 = Significant Vs. Control.Natural Product Radiance Vol 3(6) November-December 2004

Table 3: Effect of the extracts of Lawsonia inermis on

the breaking strength of incision wounds

Code given

Oral Treatment

Control

IA 125.0±12.1

Petroleum ether (40-60°) extract

B196.7±10.7*

CWorofonn extract

C145.1±6.0

Ethanol extract

D213.0±12.6*

Aqueous extract

E178.0±7.2*

Lawsone

F216.0±12.5*

Topical Treatment Control (simple ointment base USP)

G126.0±6.3

Ethanol extract ointment (30% w/w)

H445.0±7.5*

Lawsone ointment (0.1 % w/w)

I414.0±6.2*

All values are given in Mean±S.E.; *p<0.05 = Significant Vs. Control.

Table 4: Comparison between topical application of ethanolextract ointment and Lawsone ointment

-;.;;;:. ..: -. ~ . 0.'·'.·' '~""o··m' ..'w' .."O"" "00'0

Excision Incision--

"A ,- Period ofSize of scar areaBJ;eaking strengthParameters

.~ .(mmZ)(g)on 4

1i

dayl2 (days)

Control (simple

ointment base USP)

77.6±1.1722.6±0.5038.0±2.1126.0±6.3

Ethanol extract ointment (30% w/w)

99.7±0.10*16.4±0.4S*12.3±0.66*444.0±7.5*

Lawsone ointment l 414.0±6:2*(0.1% w/w) 99.4±0.12*17.8±0.36*14.5±1.42*

~

-- - --- -----______ l.-_

All values are given in Mean±S.E.; *p<0.05= Significant Vs. Control.

Natural Product Radiance Vol 3(6) November-December 2004

Ar,;,I.

7. Trease GEand Evans WC,Extraction

of plant material, separation andisolation of constituents, In:Pharmacognosy, 14th edn, W.B.

Saunders Company, London, 1997,248.

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healing Profiles, Indian Drugs,1990,27,342.

12. Ghosh MN, Calculation ofLD50 and itserror, In: Fundamentals of

Experimental Pharmacology,

Scientific Book Agency, Calcutta,1971,112.

13. Paget GE and Barnes JM, In:

Pharmacometrics, EvaluationofDrugActivities, edited by DR Laurence,

Academic Press, New York, Vol. I,

1983,115.

14. Morton JJP and Malone MH,Effectofcortisone and anabolic steroids on the

tensile strength of healing wound,

Arch Int Pharmacodyn, 1972,196,117.

15. Ehrlich HP and Hunt TK,Evaluation

of voluntary activityby a open woundprocedure, Ann Surg, 1969, 170,203.

16. Lee KH,Studies on the mechanism ofaction II. Retardation of wound

healing by aspirin, J Pharm Sci,1968, 57, 1042.

17. BairyKLand Rao CM,Wound healing

profiles of Gingko biloba, J NatRemed, 2001, 1, 25.

by AK Nadkarni, 1954, Vol. I,

reprinted, 1999, 730.

Pharmacographica Indica: A History

ofPrinciple Drugs ofVegetableOrigin,

met with in British India, by W

Dymock, CJHWarden and D Hooper,

3 Vols,1889-1893, reprinted (Bishen

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Ingredients Used In Food, Drugs And

Cosmetics, '2nd edn, A Wiley­

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the immuno-stimulant activity ofNapthoquinone extract of leaves of

Lawsonia alba, Indian Drugs,1998, 35, 427.

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drugs, In: Practical Pharmacognosy,4th edn, Vallabh prakashan, Delhi,

1994, 146.

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Plants, by RP Rastogi and BN

Mehrotra (Central Drug ResearchInstitute, Lucknowand Publication &

Information Directorate, NewDelhi),

Vol.III, 1993, 385.

Conclusion

The results of the present studyhave led to the conclusion that 4.

transdermal drug delivery systemcontaining ethanol extracts has exhibited

more prominent wound healing activitythan trans dermal drug delivery system

containing other extracts and also withisolated lawsone formulation, when given

by topical route. Similarlyethanol extract

and isolated lawsone were also 5significantly better than other extracts .

when given by oral route. This improvedwound promoting activity of ethanolextract could be attributed to the

additional presence oflawsone along withthe flavonoids that as alluded earlier have

significantwound healing activity.Further 6.in our studies, itwas found that the topicalapplication of ethanol extract as well asisolated lawsone was more effectivethan

the same given by the oral route.

References

2. BairyKL,Wound healing potentials of

some plant products, J Nat Remed,2002,2, 11.

1. UdupaSL,UdupaALand KulkarniDR,

Effect of Tridax procumbensextract on wound healing, PlantaMed, 1991, 57, 325.

3. Indian Materia Medica, by KM

Nadkarni (Popular Book Depot,Bombay & Dhootapapeshwar 9.

Prakashan Ltd, Panvel), Lawsoniaalba Lam.,L. spinosa; L. inermis,Linn., 3rd edn, revised and enlarged

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18. Narayana KR, Reddy SM, Chaluvadi

MR and Krishna DR, Bioflavonoids

classification, pharmacological,

biochemical effects and therapeutic

potential, Indian J Pharmacol,2001,33,2.

19. Robak] and Glyglewski R], Evaluation

of wound healing activity of selected

traditional medicinal plants from

Peru, Biochem Pharmacol, 1988,37,837.

20. Padmaja PN, Bairy KL and Kulkarni

DR, Pro-healing effect of betel nut and

its polyphenols, Fitoterapia, 1994,65(4), 298-300.

Applications of Henna in Unani Systemsof Medicine

In Unani practices Henna (Lawsonia inermis Linn.) leaves, flowers and seeds are used in various diseases

except respiratory disorders (due to its adverse effect on lungs). Some of its uses are given below:

1. For the treatment ofleprosy in primary stage supernatant fluid (100 ml) ofleaves (420 g soaked in water over night)

mixed with 35 g sugar is taken daily for 20 days in the. morning.

2. Leaves are gr6und with Sutra nium (Orchis sp.) to make a paste and applied on hair. It gives very dark red

colour and protects hair falling.

3. Paste of leaves is applied on rough hands to make them soft.

4. Paste of leaves prepared in hot water, protects pustule formation of small pox and from skin marks.

5. Leaves alone or with Kishniz (Coriandrum sativum Linn.) are boiled in water and used as medicated footbath

or irrigation on burns and scald wounds and their marks. This preparation is also used for the treatment of gingivi­

tis, stomatitis and excessive salivation.

6. Leaves are ground and mixed with rose oil to use locally to cure scabies and psoriasis.

7. Leaves ground with leaves of A rand (Ricinus communis Linn.) are used locally to fill kracked heel.

8. Leaves are used as substitute for Socotra Dragon' Blood Tree (Dracaena cinnabari Balf.f.) as antihaemorrhagic

agent. Leaves (12 g) and Gentian (Gentiana lutea Linn.) (24 g) are ground and mixed with water to make paste

and applied on hand and foot as styptic in excessive bleeding during lochia period.

9. Leaves ground with fat of goat milk are applied on nails as corrective to wounds and deformities of nails.

10. Leaves are ground widl Khall (Vinegar) to make paste and apply on skull as effective remedy for migraine and

epiphora.

12. Leaves (250 g) are boiled in water and supernatant fluid is used orally as effective medicine for jaundice, hepatitis,

nephrolithiasis, urolithiasis, dysurea and dysmenorrhoea. The fluid is also used in tub bath to cure all types of

uterine pain.

13. Leaves (4.5 g) paste mixed with honey is used orally to cure all type of headache and nasal secretion as rhinitis,

epitasis, etc.

14. Flower oil is used locally as complexion enhancer, rubefacient, resolvent for all types of abscess, hair grower and innerves weakness.

15. Seeds (4.5 g) ground and mixed with honey are used orally to give relief in high blood pressure and brain disorders

(Contributed by: Dr. Mohd. Danish Mal1fooz, TKDL,NISCAIR,New Delhi)

Natural Product Radiance Vol 3(6) November-December 2004